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Microbial genetics
Genetics DNA, its manipulation and the consequences for the organism.Aims to understand cellular functions and properties of organisms.
classical genetics: Phenotype genotype proteinMutant-Analysis, Genotype-Phenotype correlation, crosses, gene transfer
molecular genetics: Isolate und Analyse DNA, cloning, in vitro Mutagenesis
“Reverse genetics”:Protein Genephenotypeconstruction of mutants; analyze in vivo function („phenotype“)
prokaryotic eukaryoticmolecular biology
central dogma of molecular biology:
Structure of bacterial DNA- chemical structure: A, C, G, T (like eukaryotes, exception: unmethylated CpG)-„free“ state (not enclosed by a membrane)- nucleoid ≙ eukaryotic chromosome
„nucleoid“
Properties of DNA- 1 bp = 0.34 nm (along helix axis)- 1 helical turn = 10.4 bp- 1 nucleotide = 330 Da, 1 bp = 660 Da- 100 nt: fairly rigid- chromosomal DNA: flexible- resistant to alkaline treatment
UV absorption spectrum: -1 OD260 double stranded DNA = 50 μg/ml-1 OD260 single stranded DNA = 33 μg/ml
-
- -
- - - -
Forces:-Effective charge of the molecule-Electrical field strength-cancelled by Friction (stokes law)-gel matrix = sieve Small DNA molecules migrate faster
-
+
Agarose gel electrophoresis
Stokes law: F = 6πrην
F = frictional force
r = particle radius
η = fluid viscosity
v = particles speed
Working with DNAC. Denaturation/Hybridization
Tm = 81.5 + 16.6(log10[Na+]) + 0.41(fraction G+C) -600/N
„probe“
„Southern“ hybridization
DNA topologyDefinition: knot-like arrangements that segments of DNA may assume.
positive supercoil: twisted in same direction as right handed helixnegative supercoil: DNA twisted opposite to hight handed helix
DNA topology: requires constraints
„Relaxed“ DNA supercoiled DNA constraint
Proteins
circular DNA
DNA topoisomerasesTopoisomerase I
nicks one strand of DNA double helixrelaxes 1 negative supercoil per nickimportance: in front of replication fork
Topoisomerase IIcuts both strands of DNA double helixrelaxes or introduces 2 supercoils per cutimportance: DNA gyrase from E. coli (replication, decatenation)
supercoiled plasmids
non-denaturing agarose gel-electrophoresisstaining of DNA: ethidium bromide
plasmid (neg. supercoiled)
denatured supercoiled(non-digestible)
plasmid (relaxed)
-
+
chromosomal DNA(contamination)
-R +R -R +R
plasmid multimers
DNA topology (Bacteria, Archaea)
chinolones (i.e. nalidixic acid)fluorochinolones (i.e. ciprofloxacin)novobiocin
Gyrase inhibitors = antibiotics:
Ciprobay
Bacterial genome:
• Size: 5 x 105 – 107 bp
• Chromosome(s)– mostly haploid (generally one chromosome)– mostly circular– supercoiled– organized in nucleoid (1 histone like protein/100 bp)– contains essential genes (+ non-essential genes, mobile
elements)
• Plasmids (facultative)
• Phages (facultative)
Genomics = analyzing the genome
• Genetic mapping: markers (conjugation, transduction)
• Physical map: restriction map (size; #chromosomes, linear/circular)
• high resolution map: clone overlapping DNA fragments
• sequencing: ordered library of plasmidsshotgun
physical map of R. sphaeroides
Genome sequencing
1977 Bacteriophage FX174 5 kb1982 Phage l 48 kb1990 Vaccinia 192 kb1991 Cytomegalovirus 229 kb1992 Marchantia polymorpha Mitochondrium 187 kb1992 Marchantia polymorpha Chloroplast 121 kb1993 Variola (Pocken) 186 kb1995 Haemophilus influenzae Rd 1830 kb1995 Mycoplasma genitalium 580 kb1996 Saccharomyces cerevisiae 12’500 kb1996 Mycoplasma pneumoniae 816 kb1996 Methanococcus jannaschii 1665 kb1996 Synechocystis PCC6803 3573 kb1997 Escherichia coli 4639 kb1997 Bacillus subtilis 4200 kb1997 Helicobacter pylori 1668 kb
Chromosome organization
Prokaryotes: few introns, little repetitive DNA (Alu etc)Sequence database: http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi
(141 on 8.4.02)
(15 on 8.4.02)
(41 finished + unfinished on 8.4.02)
Science. 1997 277(5331):1453-74.
rrn operons
tRNA genes
REP sequences
G/C composition of +/- strand
phage proteins
E. coli K12 chromosome
4.7 x 106 bp, circular
4288 genes; 38% unknown
7 rRNA operons
86 tRNA genes
2192 transcriptional units, 73% monocistronic; 6% >4 genes
start codons: 83% ATG; 14% GTG; 3% TTG; 1 x ATT; 1 x CTG
405 genes with start/stop overlap
largest orf = 2383 aa protein
average orf = 317 aa
314 rep elements
E. coli chromosome
bacterial genomics
genome comparison:
conserved ORF (minimal genome??)
mechanism of genome expansion/contraction
evolution of bacteria (commensal pathogen)
genome analysis:
metabolic functions
prediction of nutrient requirements
study characteristics
Evolution of bacterial genomesmutations
Rearrangements, deletions, horizontal gene transfer(Lawrence (1997) Trends Microbiol. 5: 355-359)
Original chromosome
4800 kb 100 Million years
Final chromosome
4800 kb
3000 kb new DNA
620 kb „old“ DNAdeleted
2380 kb of new DNAlost right away
620 kb new
DNA integratedE. coli(old)
E. coli(new)
Minimal gene set
- metabolites can be imported; proteins not relies on own gene set
Minimal gene set = „least # of genes required for life in extremely rich media“
experimental:transposon mutagenesisdeletion of each single orf
computational:small genomes of parasites/endosymbionts
(Mycoplasma, Buchnera)conserved genes between small genomes (… 150-300 ?)
….there may not be a singular exact answer!!!
Minimal number of protein genes: 470 (Mycoplasma genitalium)
Genome size: 4.6 Mbp 1.8 Mbp 0.6 Mbp
RUSSELL F. DOOLITTLE Nature 416, 697 - 700 (2002)
Genes „lost“ in the smallest genomes
bacteria living inside host cells
a „minimal metabolism“
Microbiol Mol Biol Rev. 2004 Sep;68(3):518-37
genome: prediction of metabolic pathwaysHelicobacter pylori 26695circular genome1,667,867 bpNature 388, 1997, S. 545
Nature 388, 1997, S. 545
H. pylori: prediction of metabolic capacity
Analysis of „non-cultivatable“ bacteria
<< 5 % of all bacteria have been cultured
bacterial consortia in nature:
soil
gut
water
genomics approach:
DNA isolation + sequence analysis (Cosmids, shotgun)
sequence assembly
prediction of metabolic capacity
study composition of consortium
design appropriate culture media
Genome based design of Media for Tropheryma whipplei
in total: 9 aa biosynthesis pathways missing7 aa biosynthesis pathways incomplete
T. whipplei cultivated in fibroblast cell line (HEL)
axenic medium: DMEM (aa), 10% FCS,1% glut., non-essential aa
The Lancet (2003), 362, pp. 447-449
Bdellovibrio bacteriovorus
Bdellovibrio bacteriovorus3.7 Mbp50% GC3584 Orfsmany: DNases, proteases, RNases, glycanases, lipases
A Predator Unmasked: Life Cycle of Bdellovibrio bacteriovorus from a Genomic PerspectiveSnjezana Rendulic et al., Science Jan 30 2004: 689-692.
Bdellovibrio bacteriovorus: hydrolytic enzymes
Bacterial genome:• Size: 5 x 105 – 107 bp
• Chromosome(s)– mostly haploid (generally one chromosome)– mostly circular– supercoiled– organized in nucleoid (1 histone like protein/100 bp)– contains essential genes (+ non-essential genes, mobile
elements)
• Plasmids (facultative)
• Phages (facultative)
Plasmids
no „house keeping“/essential genes
- size: 1 – 1000 kb (< 5% of chrom.)
- double stranded DNA
- supercoiled
- 1 to >100 copies / cell
- autonomous replication („replicon“)
-replication controlled by feedback-loops (plasmid/host factors)
- very abundant in nature:300 identified in E. coli isolates
Plasmids
Nomenclature for recombinant Plasmids:
pXY000
XY: shorthand for name of researcher
000: continuous numbering
zB. pBR322 is the 322th plasmid
made by Bolivar and Rodriguez.
paramters of interest: example: cloning vector
size small
copy number high (10->100)
selection marker antibiotic resistance
host range narrow (safety)
fertility (conjugation?) non conjugative
ori of replication (incompatibility group) i.e. ColE1
additional genes i.e. lacZ- (insert screening)
Plasmid encoded phenotypes
Examples of naturally ocurring plasmids
F-Plasmid Fertility plasmid for conjugative transfer of genes
R-Plasmids Antibiotics resistancee.g. amp, kan, tet, cam
ColE1 Production von colicin, a bacteriocin against E. coli
Ti Tumor initiation in plants
pSym symbiotc plasmid for nodulation and N2-fixation
Tol degradation of toluene
E. coli F-plasmid
99,159 bp
replication + segregation
LactococcusplasmidpK214
29871 bp
5000
10000
15000
20000
25000
inv soj 03orf59
orf60 orf61
repB
orf63
oriT
nel
IS904
mef214
phnBp35
IS904#IS215binL
rob
IS214-III
str
repDnick site
mob
cat
rep#
IS214-II
orf7 (Tn916)
orf9 (Tn916)
27
tetS
IS214-I
ChloramphenicolR
tetracyclinR
streptomycinR
replication:repB + iterons
macrolideefflux pump
ChloramphenicolR
tetracyclinR
streptomycinR
replication:repB + iterons
macrolideefflux pump
Overview:
2 ways to acquire resistance:mutation of a target geneacqusition of novel genes (source antibiotic producers)
genetic basis of spread of antibiotics resistancefactors involved in spread of resistance:
medicineagriculture
Antibiotic resistance in pathogenic bacteria
Where does antibiotic resistance come from?
plasmids = tools in modern molecular biology
vector property_______________________
Cloning: obtain/analyze a specific piece of DNA
Expression: regulated promotor
Shuttle: promotors/ori for diverse organisms
Mobilizable: transferable by conjugation
broad host range: works in diverse bacteria
suicide: site directed mutagenesis
cosmids: plasmid/phage lambda etc.
pBR322
ancestor of most general cloning vectors:
pGEX-3X
Bacterial genome:
• Size: 5 x 105 – 107 bp
• Chromosome(s)– mostly haploid (generally one chromosome)– mostly circular– supercoiled– organized in nucleoid (1 histone like protein/100 bp)– contains essential genes (+ non-essential genes, mobile
elements)
• Plasmids (facultative)
• Phages (facultative)
Bacteriophages =
bacterial viruses
Bacteriophages: plaque formation
Virulent phages
Temperate phages
Phage lambda genome
E. coli O157 Sakai
LEE-locus(Type III secr.)
stx1
stx2
E. coli K12 vs. O157 Sakai
LEE-locus
stx2
stx2
Phage „cargo“ genes
Evolution of bacterial genomesMutations
Rearrangements, deletions, horizontal gene transfer(Lawrence (1997) Trends Microbiol. 5: 355-359)
Original chromosome
4800 kb 100 Million years
Final chromosome
4800 kb
naked DNAplasmidsphages
620 kb „old“ DNA(includes plasmids, phages)
includes:plasmidsphages
620 kb new
DNA integratedE. coli(old)
E. coli(new)